Downregulation of miR-144 is associated with colorectal cancer progression via activation of mTOR signaling pathway

TakeshiIwaya 1 2 Takehiko Yokobori 0 Naohiro Nishida 2 Ryunosuke Kogo 2 Tomoya Sudo 2 Fumiaki Tanaka 2 Kohei Shibata 2 Genta Sawada 2 Yusuke Takahashi 2 Masahisa Ishibashi 2 Go Wakabayashi 1 Masaki Mori 3 Koshi Mimori 2 0 Department of General Surgical Science, Graduate School of Medicine, Gunma University , 3-39- 22 Showa-machi, Maebashi, Gunma 371-8511, Japan 1 Department of Surgery, Iwate Medical University , Morioka 020-8505, Japan 2 Department of Surgery, Kyushu University Beppu Hospital , 4546 Tsurumihara, Beppu 874-0838, Japan 3 Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University , 2-2 Yamadaoka, Suita 565-0871, Japan The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: - *To whom correspondence should be addressed. Tel:+81 977 1650; Fax: +81 977 1651; Email: The mammalian target of rapamycin (mTOR) is a downstream integrator of essential pathways. mTOR signaling is frequently dysregulated in a variety of human cancers, and in silico analysis has revealed two miR-144 binding sites in the mTOR 3 untranslated region. We investigated the clinicopathologic magnitude of the mTOR pathway regulating microRNA, miR-144 in colorectal cancer (CRC) cases. The regulation of mTOR by miR-144 was examined with inhibitor miR-144-transfected cells. We also investigated changes in sensitivity to the mTOR inhibitor, rapamycin, in inhibitor miR-144-transfected cells. Quantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-144 expression in 137 CRC. Furthermore, we assessed the correlation between CRC prognosis and the expression of 16 genes in the Akt/mTOR pathway. In vitro assays showed that mTOR is a direct target of miR-144, and downregulation of miR-144 facilitated proliferation of CRC cell line, HT29. In addition, the viability of HT29 cells with downregulated miR-144 expression was significantly reduced with rapamycin treatment. Low expression levels of miR-144 were associated with enhanced malignant potential such as venous invasion (P=0.0013), liver metastasis (P=0.08), liver recurrence (P = 0.0058) and poor prognosis (P = 0.0041). Multivariate analysis indicated that low miR-144 expression was an independent prognostic factor for survival. Among many genes consisting of the mTOR pathway, only high expression of Rictor was associated with poor prognosis of CRC. miR-144 is a meaningful prognostic marker. Downregulation of miR-144 leads to poor prognosis of CRC patients via activation of the mTOR signaling pathway. Introduction The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism and the cytoskeleton, and its signaling pathway is frequently dysregulated in a variety of human cancers (1,2). Rapamycin and rapamycin derivatives have long been employed for immunosuppression and, more recently, as anticancer treatments. The drug is now clinically used for the treatment of advanced renal cell cancer, and Phase III trials are under way for breast cancer, gastric cancer, hepatocellular Abbreviations: CI, confidence interval; CRC, colorectal cancers; LMD, laser microdissection; miRNAs, micro RNAs; mTOR, mammalian target of rapamycin; RR, relative risk; UPL, Universal Probe Library Probe; 3UTR, 3 untranslated region. These authors contributed equally to thiswork. carcinoma, pancreatic neuroendocrine tumors and lymphoma, following favorable results of Phase II studies (38). Since 60% of colorectal cancers (CRC) exhibit high levels of activated Akt (9), the association between mTOR and CRC has also been investigated. It has been reported that mTOR was highly activated in glandular elements of CRC and in colorectal adenomas with highgrade intraepithelial neoplasia, with a correlation between immunohistochemical staining intensity and depth of infiltration (10). mTOR exists in two functionally distinct complexes: mTORC1 (containing mTOR, Raptor, etc.) and mTORC2 (containing mTOR, Rictor, etc.). Gulhati et al. demonstrated increased expression of mTOR, Raptor and Rictor mRNA in more advanced stages of CRC. In addition, mTOR, Raptor and Rictor protein levels were significantly elevated in stage IV CRCs (11). These clinical observations have led to the association of higher grade CRC malignancies with increased expression of mTOR and its complexes. Activation of Akt/mTOR signaling is also frequently observed in CRCs, and mTOR inhibitors have an anti-proliferative effect in several CRC cell lines (11,12). Therefore, mTOR kinase inhibitors, such as rapamycin, have been investigated as part of the therapeutic regimen for CRC patients. In order to predict the efficacy of rapamycin for CRC patients, it is important to know the value of mTOR expression status as a prognostic or susceptibility marker in clinical settings ofCRC. Micro RNAs (miRNAs) are 19- to 25mer non-coding RNAs that incompletely bind the 3 untranslated region (UTR) of multiple target mRNAs, enhancing their degradation and inhibiting translation. miRNAs possess normal biological functions, such as regulation of proliferation, differentiation and apoptosis. Moreover, dysregulation of miRNAs plays a critical role in carcinogenesis and cancer progression (13). Many miRNAs are present at lower levels in cancer tissue than in normal tissue, a state that contributes to cancer progression (14). In silico analysis of miRNA-target mRNA prediction algorithm (TargetScan 6.0; http://www.targetscan.org/) revealed two miR-144 binding sites in the mTOR 3UTR region with perfect WatsonCrick matches at miRNA positions 17 and 28 (Supplementary Figure1A, available at Carcinogenesis Online). These sites raise the possibility that miR-144 is involved in the mTOR signaling pathway. In this study, we evaluated miR-144 expression status, its clinical significance in CRC and the association between miR-144 and the mTOR pathway. Materials and methods Experimental studies Cell lines and cell culture. The human colorectal cancer cell lines, HT29 and CaR-1, were obtained from the American Type Culture Collection and the Japanese Collection of Research Bioresources. These cell lines were maintained in RPMI 1640 containing 10% fetal bovine serum with 100 U/ml penicillin and 100 U/ml streptomycin sulfates and cultured in a humidified 5% CO2 incubator at 37C. Evaluation of mTOR mRNA expression in colorectal cancer cells. For RNA analysis, each cell line was seeded at 1 105 cells per well in a volume of 2 ml in 6-well flat-bottomed microtiter plates. Total RNA from these cell lines was isolated using the mirVana miRNA Isolation Kit (Ambion) following 48 h incubation. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was performed with the Universal Probe Library Probe (UPL; Roche Diagnostics) to measure mTOR mRNA expression. Primer sequences corresponding to UPL and RT-PCR protocols were described previously (15). Transfection of miR-144 inhibi (...truncated)